Chimeric immunogens

ABSTRACT

Multimeric hybrid genes encoding the corresponding chimeric protein comprise a gene sequence coding for an antigenic region of a protein from a first pathogen linked to a gene sequence coding for an antigenic region of a protein from a second pathogen. The pathogens particularly are parainfluenza virus (PIV) and respiratory syncytial virus (RSV). A single recombinant immunogen is capable of protecting infants and similar susceptible individuals against diseases caused by both PIV and RSV.

This is a divisional of application Ser. No. 08/001,554 filed Jan. 6,1993.

FIELD OF INVENTION

The present invention relates to the engineering and expression ofmultimeric hybrid genes containing sequences from the gene coding forimmunogenic proteins or protein fragments of numerous pathogens.

BACKGROUND TO THE INVENTION

The advantage of the approach taken by the present invention is toproduce single immunogens containing protective antigens from a range ofpathogens. Such chimeras greatly simplify the development of combinationvaccines, in particular, with the view ultimately to produce single dosemultivalent vaccines. Multivalent vaccines are currently made byseparately producing pathogens and/or their pertinent antigens andcombining them in various formulations. This is a labour intensive,costly and complex manufacturing procedure. In contrast, theavailability of a single immunogen capable of protecting against a rangeof diseases would solve many of the problems of multivalent vaccineproduction. Several chimeric immunogens of the type provided herein maybe combined to decrease the number of individual antigens required in amultivalent vaccine.

Human Parainfluenza virus types 1,2,3 and Respiratory syncytial virustypes A and B are the major viral pathogens responsible for causingsevere respiratory tract infections in infants and young children. It isestimated that, in the United States alone, approximately 1.6 millioninfants under one year of age will have a clinically significant RSVinfection each year and an additional 1.4 million infants will beinfected with PIV-3. Approximately 4000 infants less than one year ofage in the United States die each year from complications arising fromsevere respiratory tract disease caused by infection with RSV and PIV-3.The WHO and NIALD vaccine advisory committees ranked RSV number twobehind HIV for vaccine development while the preparation of anefficacious PIV-3 vaccine is ranked in the top ten vaccines considered apriority for vaccine development.

Safe and effective vaccines for protecting infants against these viralinfections are not available and are urgently required. Clinical trialshave shown that formaldehyde-inactivated and live-attenuated viralvaccines failed to adequately protect vaccinees against theseinfections. In fact, infants who received the formalin-inactivated RSVvaccine developed more serious lower respiratory tract disease duringsubsequent natural RSV infection than did the control group. [Am. J.Epidemiology 89, 1969, p.405-421; J. Inf. Dis. 145, 1982, p.311-319].Furthermore, RSV glycoproteins purified by immunoaffinity chromatographyusing elution at acid pH induced immunopotentiation in cotton rats.[Vaccine, 10(7), 1992, p.475-484]. The development of efficacious PIV-3and RSV vaccines which do not cause exacerbated pulmonary disease invaccinees following injection with wild-type virus would havesignificant therapeutic implications. It is anticipated that thedevelopment of a single recombinant immunogen capable of simultaneouslyprotecting infants against diseases caused by infection with bothParainfluenza and Respiratory syncytial viruses could significantlyreduce the morbidity and mortality caused by these viral infections.

It has been reported that a protective response against PIV-3 and RSV iscontingent on the induction of neutralizing antibodies against the majorviral surface glycoproteins. For PIV, these protective immunogens arethe HN protein which has a molecular weight of 72 kDa and possesses bothhemagglutination and neuraminidase activities and the fusion (F)protein, which has a molecular weight of 65 kDa and which is responsiblefor both fusion of the virus to the host cell membrane and cell-to-cellspread of the virus. For RSV, the two major immunogenic proteins are the80 to 90 kDa G glycoprotein and the 70 kDa fusion (F) protein. The G andF proteins are thought to be functionally analogous to the PIV HN and Fproteins, respectively. The PIV and RSV F glycoproteins are synthesizedas inactive precursors (FO) which are proteolytically cleaved intoN-terminal F2 and C-terminal F1 fragments which remain linked bydisulphide bonds.

Recombinant surface glycoproteins from PIV and RSV have beenindividually expressed in insect cells using the baculovirus system [Rayet al., (1989), Virus Research, 12: 169-180; Coelingh et al., (1987),Virology, 160: 465-472; Wathen et al., (1989), J. of Inf. Dis. 159:253-263] as well as in mammalian cells infected with recombinantpoxviruses [Spriggs, et al., (1987), J. Virol. 61: 3416-3423; Stott etal., (1987), J. Virol. 61: 3855-3861]. Recombinant antigens produced inthese systems were found to protect immunized cotton rats against livevirus challenge. More recently, hybrid RSV F-G [Wathan et al., (1989),J. Gen Virol. 70: 2625-2635; Wathen, published International Patentapplication WO 89/05823] and PIV-3 F-HN [Wathen, published InternationalPatent Application WO 89/10405], recombinant antigens have beenengineered and produced in mammalian and insect cells. The RSV F-Ghybrid antigen was shown to be protective in cotton rats [Wathan et al.,(1989), J. Gen. Virol. 70: 2637-2644] although it elicited a poor anti-Gantibody response [Connors et al., (1992), Vaccine 10: 475-484]. Theprotective ability of the PIV-3 F-HN protein was not reported in thepublished patent application. These antigens were engineered with theaim to protect against only the homologous virus, that is either RSV orPIV-3. However, it would be advantageous and economical to engineer andproduce a single recombinant immunogen containing at least oneprotective antigen from each virus in order simultaneously to protectinfants and young children against both PIV and RSV infections. Thechimeric proteins provided herein for such purpose also may beadministered to pregnant women or women of child bearing age tostimulate maternal antibodies to both PIV and RSV. In addition, thevaccine also may be administered to other susceptible individuals, suchas the elderly.

SUMMARY OF INVENTION

In its broadest aspect, the present invention provides a multimerichybrid gene, comprising a gene sequence coding for an immunogenic regionof a protein from a first pathogen linked to a gene sequence coding foran immunogenic region of a protein from a second pathogen and to achimeric protein encoded by such multimeric hybrid gene. Such chimericprotein comprises an immunogenic region of a protein from a firstpathogen linked to an immunogenic region of a protein from a secondpathogen.

The first and second pathogens are selected from bacterial and viralpathogens and, in one embodiment, may both be viral pathogens.Preferably, the first and second pathogens are selected from thosecausing different respiratory tract diseases, which may be upper andlower respiratory tract diseases. In a preferred embodiment, the firstpathogen is parainfluenza virus and the second pathogen is respiratorysyncytial virus. The PIV protein particularly is selected from PIV-3 Fand HN proteins and the RSV protein particularly is selected from RSV Gand F proteins. Another aspect of the invention provides cellscontaining the multimeric hybrid gene for expression of a chimericprotein encoded by the gene. Such cells may be bacterial cells,mammalian cells, insect cells, yeast cells or fungal cells. Further, thepresent invention provides a live vector for antigen delivery containingthe multimeric hybrid gene, which may be a viral vector or a bacterialvector, and a physiologically-acceptable carrier therefor. Such livevector may form the active component of a vaccine against diseasescaused by multiple pathogenic infections. Such vaccine may be formulatedto be administered in an injectable form, intranasally or orally.

In an additional aspect of the present invention, there is provided aprocess for the preparation of a chimeric protein, which comprisesisolating a gene sequence coding for an antigenic region of a proteinfrom a first pathogen; isolating a gene sequence coding for an antigenicregion of a protein from a second pathogen; linking the gene sequencesto form a multimeric hybrid gene; and expressing the multimeric hybridgene in a cellular expression system. The first and second pathogens areselected from bacterial and viral pathogens. Such cellular expressionsystem may be provided by bacterial cells, mammalian cells, insectcells, yeast cells or fungal cells. The chimeric protein product of geneexpression may be separated from a culture of the cellular expressionsystem and purified.

The present invention further includes a vaccine against diseases causedby multiple pathogen infections, comprising the chimeric protein encodedby the multimeric hybrid gene and a physiologically-acceptable carriertherefor. Such vaccine may be formulated to be administered in aninjectable form, intranasally or orally.

The vaccines provided herein may be used to immunize a host againstdisease caused by multiple pathogenic infections, particularly thosecaused by a parainfluenza virus and respiratory syncytial virus, byadministering an effective amount of the vaccine to the host. As notedabove, for human PIV and RSV , the host may be infants and youngchildren, pregnant women as well as those of a child-bearing age, andother susceptible persons, such as the elderly.

The chimeric protein provided herein also may be used as a diagnosticreagent for detecting infection by a plurality of different pathogens ina host, using a suitable assaying procedure.

It will be appreciated that, while the description of the presentinvention which follows focuses mainly on a chimeric molecule which iseffective for immunization against diseases caused by infection by PIVand RSV, nevertheless the invention provided herein broadly extends toany chimeric protein which is effected for immunization against diseasescaused by a plurality of pathogens, comprising an antigen from each ofthe pathogens linked in a single molecule, as well as to genes codingfor such chimeric molecules.

In this application, by the term “multimeric hybrid genes” we mean genesencoding antigenic regions of proteins from different pathogens and bythe term “chimeric proteins” we mean immunogens containing antigenicregions from proteins from different pathogens.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A to 1E shows the nucleotide (SEQ ID No: 1) and amino acid (SEQID No: 2) sequence of a PCR-amplified PIV-3 F gene and F protein,respectively;

FIG. 2 shows the restriction map of the PIV-3 F gene;

FIGS. 3A to 3E shows the nucleotide (SEQ ID No: 3) and amino acid (SEQID No: 4) sequences of the PIV-3 HN gene and HN protein, respectively;

FIG. 4 shows the restriction map of the PIV-3 HN gene;

FIGS. 5A to 5E shows the nucleotide (SEQ ID No: 5) and amino acid (SEQID No: 6) sequences of the RSV F gene and RSV F protein, respectively;

FIG. 6 shows the restriction map of the RSV F gene;

FIG. 7 shows the nucleotide (SEQ ID No: 7) and amino acid (SEQ ID No: 8)sequences of the RSV G gene and RSV G protein, respectively;

FIG. 8 shows the restriction map of the RSV G gene;

FIG. 9 shows the steps involved in the construction of an expressionvector containing a chimeric F_(PIV-3)-F_(RSV) gene;

FIG. 10 shows the steps involved in the construction of an expressionvector containing a F_(PIV-3) gene lacking the 5′-untranslated sequenceand transmembrane anchor and cytoplasmic tail coding regions;

FIG. 11 shows the steps involved in the construction of an expressionvector containing a chimeric F_(PIV-3)-F_(RSV) gene containing atruncated PIV-3 F gene devoid of 5′-untranslated region linked to atruncated RSV F1 gene;

FIG. 12 shows the steps involved in construction of a modified pAC 610baculovirus expression vector containing a chimeric F_(PIV-3)-F_(RSV)gene consisting of the PIV-3 F gene lacking both the 5′-untranslatedsequence as well as transmembrane and cytoplasmic tail coding regionlinked to the truncated RSV F1 gene;

FIGS. 13A and 13B show immunoblots of cell lysates from Sf9 cellsinfected with recombinant baculoviruses;

FIG. 14 shows the steps involved in constructing a baculovirus transfervector (pD2);

FIG. 15 shows the steps involved in construction of a chimericF_(RSV)-HN_(PIV-3) gene;

FIGS. 16A and 16B show an SDS-PAGE gel and immunoblot of purifiedF_(RSV)-HN_(PIV-3) chimeric protein;

FIG. 17 illustrates mutagenesis of a PIV-3 F gene; and

FIG. 18 shows the steps involved in the construction of a chimericF_(PIV-3)-G_(RSV) gene.

GENERAL DESCRIPTION OF INVENTION

In the present invention, a chimeric molecule protective against twodifferent major childhood diseases is provided. The present inventionspecifically relates to the formulation of various recombinantParainfluenza virus (PIV)/Respiratory syncytial virus (RSV) immunogensto produce safe and efficacious vaccines capable of protecting infantsand young children, as well as other susceptible individuals, againstdiseases caused by infection with both PIV and RSV. However, asdescribed above, the present invention extends to the construction ofmultimeric hybrid genes containing genes coding for protective antigensfrom many pathogens. Such vaccines may be administered in any desiredmanner, such as a readily-injectable vaccine, intranasally or orally.

In the present invention, the inventors have specifically engineeredseveral model PIV/RSV chimeric genes containing relevant sequences fromselected genes coding for PIV-3 and RSV surface glycoproteins linked intandem. All genes in the chimeric constructs described herein wereobtained from recent clinical isolates of PIV-3 and RSV. The chimericgene constructs may include gene sequences from either PIV-3 F or HNgenes linked in tandem to either RSV F or G genes in all possiblerelative orientations and combinations.

The chimeric gene constructs provided herein may consist of either theentire gene sequences or gene segments coding for immunogenic andprotective epitopes thereof. The natural nucleotide sequence of thesegenes may be modified by mutation while retaining antigenicity and suchmodifications may include the removal of putative pre-transcriptionalterminators to optimize their expression in eukaryotic cells. The geneswere designed to code for hybrid PIV-RSV surface glycoproteins linked intandem in a single construct to produce gene products which elicitprotective antibodies against both parainf luenza and respiratorysyncytial viruses. Such multimeric hybrid genes consist of a genesequence coding for a human PIV-3 F or HN protein or an immunogenicepitope-containing fragment thereof linked to a gene sequence coding fora human RSV G or F protein or an immunogenic epitope-containing fragmentthereof. Specific gene constructs which may be employed includeF_(PIV-3)-F_(RSV), F_(RSV)-HN_(PIV-3) and F_(PIV-3)-G_(RSV) hybridgenes.

In addition, the present invention also extends to the construction ofother multimeric genes, such as trimeric genes containing PIV and RSVgenes or gene segments, linked in all possible relative orientations.For example:

F_(PIV)-HN_(PIV)-F or G_(RSV)

F_(PV)-F_(RSV)-G_(RSV)

HN_(PIV)-F_(RSV)-G_(RSV)

The multimeric genes provided herein also may comprise at least one geneencoding at least one immunogenic and/or immunostimulating molecule.

The multimeric hybrid genes provided herein may be sub-cloned intoappropriate vectors for expression in cellular expression systems. Suchcellular expression systems may include bacterial, mammalian, insect andfungal, such as yeast, cells.

The chimeric proteins provided herein also may be presented to theimmune system by the use of a live vector, including live viral vectors,such as recombinant poxviruses, adenoviruses, retroviruses, SemlikiForest viruses, and live bacterial vectors, such as Salmonella andmycobacteria (e.g. BCG).

Chimeric proteins, such as a PIV/RSV chimera, present in either thesupernatants or cell lysates of transfected, transformed or infectedcells then can be purified in any convenient manner.

To evaluate the immunogenicity and protective ability of the chimericproteins, suitable experimental animals are immunized with eithervarying doses of the purified chimeric proteins, such as the PIV/RSVchimera, and/or live recombinant vectors as described above. Suchchimeric proteins may be presented to the immune system by either theuse of physiologically-acceptable vehicles, such as aluminum phosphate,or by the use of delivery systems, such as ISCOMS and liposomes. Thechimeras also may be formulated to be capable of eliciting a mucosalresponse, for example, by conjugation or association withimmunotargeting vehicles, such as the cholera toxin B subunit, or byincorporation into microparticles. The vaccines may further comprisemeans for delivering the multimeric protein specifically to cells of theimmune system, such as toxin molecules or antibodies. To further enhancethe immunoprotective ability of the chimeric proteins, they may besupplemented with other immunogenic and/or immunostimulating molecules.The chimeric PIV/RSV proteins specifically described herein may beformulated with an adjuvant, such as aluminum phosphate, to producereadily-injectable vaccines for protection against the diseases causedby both PIV-3 and RSV. The chimeric proteins also may be administeredintranasally or orally. The chimeric proteins may be used in test kitsfor diagnosis of infection by PIV-3 and RSV.

The invention is not limited to the preparation of chimeric PIV-3 andRSV proteins, but is applicable to the production of chimeric immunogenscomposed of either the entire sequences or regions of the immunogenicproteins from at least two pathogens sequentially linked in a singlemolecule. Chimeric antigens also may be synthesized to contain theimmunodominant epitopes of several proteins from different pathogens.These chimeric antigens may be useful as vaccines or as diagnosticreagents.

Sequence Identification

Several nucleotide and amino acid sequences are referred to in thedisclosure of this application. The following table identifies thesequences and the location of the sequence:

SEQ ID No. Identification Location  1 Nucleotide sequence for FIG. 1,Example 1 PCR-amplified PIV-3 F gene  2 Amino acid sequence for FIG. 1,Example 1 PCR-amplified PIV-F protein  3 Nucleotide sequence for FIG. 3,Example 1 PIV-3 HN gene  4 Amino acid sequence for FIG. 3, Example 1PIV-3 HN protein  5 Nucleotide sequence for FIG. 5, Example 1 RSV F gene 6 Amino acid sequence for FIG. 5, Example 1 RSV F protein  7 Nucleotidesequence for FIG. 7, Example 1 RSV G gene  8 Amino acid sequence forFIG. 7, Example 1 RSV G protein  9 BsrI - BamHI oligo- FIG. 9, Example 2nucleotide cassette 10 BspHI - BamHI oligo- FIG. 9, Example 2 nucleotidecassette 11 EcoRI - Ppu MI oligo- FIG. 9, Example 2 nucleotide cassette12 BrsI - BamHI oligo- FIG. 10, Example 3 nucleotide cassette 13 EcoRI -Bsr BI oligo- FIG. 10, Example 3 nucleotide cassette 14 EcoRV - EcoRIoligo- FIG. 11, Example 5 nucleotide cassette 15 EcoRV - BamHI oligo-FIG. 14, Example 8 nucleotide cassette 16 BspHI - BspHI oligo- FIG. 15,Example 9 nucleotide cassette 17 Nucleotide sequence for Example 15PIV-3 F gene 18 Mutagenic oligo- FIG. 17, Example 15 nucleotide #2721 19Nucleotide sequence for Example 15 part of oligo- nucleotide #2721 20oligonucleotide probe Example 15

Deposit Information

Certain plasmid DNAs described and referred to herein have beendeposited with the American Type Culture Collection (ATCC) located at10801 University Boulevard, Manassas, Va. 20110-2209, pursuant to theBudapest Treaty and prior to the filing of this application. Thedeposited purified plasmids will become available to the public and allrestrictions imposed on access to the deposits will be removed upongrant of this U.S. patent application or upon publication of itscorresponding European patent application, whichever first occurs. Theinvention described and claimed herein is not to be limited in scope bythe plasmid DNAs of the constructs deposited, since the depositedembodiment is intended only as an illustration of the invention. Thefollowing purified plasmids were deposited at the ATCC with the notedaccession numbers on Dec. 17, 1992:

Plasmid Example No. Accession No. pAC DR7 5 75387 pD2RF-HN 9 75388pD2F-G 16  75389

Any equivalent plasmids that can be used to produce equivalent antigensas described in this application are within the scope of the invention.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods for cloning and sequencing the PIV-3 and RSV genes as well asthe procedures for sub-cloning the genes into appropriate vectors andexpressing the gene constructs in mammalian and insect cells are notexplicitly described in this disclosure but are well within the scope ofthose skilled in the art.

Example 1

This Example outlines the strategy used to clone and sequence the PIV-3F, HN and RSV F, G genes (from a type A isolate). These genes were usedin the construction of the F_(PIV-3)-F_(RSV), F_(RSV)-HN_(PIV-3), andF_(PIV-3)-G_(RSV) chimeric genes detailed in Examples 2 to 4, 9 and 15,respectively.

Two PIV-3 F gene clones initially were obtained by PCR amplification ofcDNA derived from viral RNA extracted from a recent clinical isolate ofPIV-3. Two other PIV-3 F gene clones as well as the PIV-3 HN, RSV F andRSV G genes were cloned from a cDNA library prepared from mRNA isolatedfrom MRC-5 cells infected with clinical isolates of either PIV-3 or RSV(type A isolate). The PIV-3 F (both PCR amplified and non-PCRamplified), PIV-3 HN, RSV F and RSV G gene clones were sequenced by thedideoxynucleotide chain termination procedure. Sequencing of bothstrands of the genes was performed by a combination of manual andautomated sequencing.

The nucleotide (SEQ ID No: 1) and amino acid (SEQ ID No: 2) sequences ofthe PCR amplified PIV-3 F gene and F protein, respectively, arepresented in FIG. 1 and the restriction map of the gene is shown in FIG.2. Sequence analysis of the 1844 nucleotides of two PCR amplified PIV-3F gene clones confirmed that the clones were identical. Comparison ofthe coding sequence of the PCR-amplified PIV-3 F gene clone with that ofthe published PIV-3 F gene sequence revealed a 2.6% divergence in thecoding sequence between the two genes resulting in fourteen amino acidsubstitutions.

The nucleotide sequence of the non-PCR amplified PIV-3 F gene clonediffered from the PCR amplified gene clone in the following manner: thenon-PCR amplified clone had ten additional nucleotides (AGGACAAAAG) (SEQID NO: 21) at the 5′ untranslated region of the gene and differed atfour positions, 8 (T in PCR-amplified gene to C in non-PCR amplifiedgene), 512 (C in PCR-amplified gene to T in non-PCR amplified gene), 518(G in PCR-amplified gene to A in non-PCR amplified gene) and 1376 (A inPCR-amplified gene to G in non-PCR amplified gene). These changesresulted in three changes in the amino acid sequence of the F proteinencoded by the non-PCR amplified PIV-3 F gene. Serine (position 110),glycine (position 112), and aspartic acid (position 398) in the primaryamino acid sequence of the F protein encoded by the PCR amplified PIV-3F gene was changed to phenylalanine (position 110), glutamic acid(position 112) and glycine (position 398), respectively, in the primaryamino acid sequence of the F protein encoded by the PCR amplified clone.

FIG. 3 shows the nucleotide (SEQ ID No: 3) and amino acid (SEQ ID No: 4)sequences of the PIV-3 HN gene and protein, respectively and therestriction map of the gene is presented in FIG. 4. Analysis of the 1833nucleotide sequence from two HN clones confirmed that the sequences wereidentical. A 4.4% divergence in the coding sequence of the PIV-3 HN genewas noted when the sequence was compared to the published PIV-3 HNcoding sequence. This divergence resulted in seventeen amino acidsubstitutions in the amino acid sequence of the protein encoded by thePIV-3 HN gene.

The nucleotide (SEQ ID No: 5) and amino acid (SEQ ID No: 6) sequences ofthe RSV F gene and RSV F protein, respectively, are shown in FIG. 5 andthe restriction mag of the gene is shown in FIG. 6. Analysis of the 1886nucleotide sequence from two RSV F clones verified complete sequencehomology between the two clones. Comparison of this nucleotide sequencewith that reported for the RSV F gene revealed approximately 1.8%divergence in the coding sequence resulting in eleven amino acidsubstitutions.

The nucleotide (SEQ ID No: 7) and amino acid (SEQ ID No: 8) sequences ofthe RSV G gene and RSV G protein, respectively, are presented in FIG. 7while the restriction map of the gene is outlined in FIG. 8. Comparisonof the 920 nucleotide sequence of the G gene clone with the published Gsequence (type A isolate) revealed a 4.2% divergence in the nucleotidesequence and a 6.7% divergence in the amino acid sequence of the geneproduct. This divergence resulted in twenty amino acid substitutions.

The full-length PIV-3 F (non-PCR amplified), PIV-3 HN, RSV F and RSV Ggenes were cloned into λgtll and subcloned into the multiple cloningsite of a Bluescript M13-SK vector, either by blunt end ligation orusing appropriate linkers. The PCR-amplified PIV-3 F gene was directlycloned into the Bluescript vector. The cloning vectors containing thePIV-3 F-PCR amplified, PIV-3 F non-PCR amplified, PIV-3 HN, RSV F andRSV G genes were named pPI3F, pPI3Fc, pPIVHN, pRSVF and pRSVG,respectively.

Example 2

This Example illustrates the construction of a Bluescript-basedexpression vector (pMCR20) containing the chimeric F_(PIV-3)-F_(RSV)gene. This chimeric gene construct contains the 5′ untranslated regionof the PIV-3 F gene but lacks the hydrophobic anchor and cytoplasmictail coding regions of both the PIV-3 and RSV F genes. The stepsinvolved in the construction of this plasmid are summarized in FIG. 9.

To prepare the PIV-3 portion of the chimeric gene (FIG. 9, step 1), thefull length PIV-3 gene lacking the transmembrane region and cytoplasmictail coding regions was retrieved from plasmid pPI3F by cutting thepolylinker with BamHI, blunt-ending the linearized plasmid with Klenowpolymerase and cutting the gene with BsrI. A BsrI-BamHI oligonucleotidecassette (SEQ ID No: 9) containing a PpuMI site and three successivetranslational stop codons were ligated to the truncated 1.6 Kb[BamHI]-BsrI PIV-3 F gene fragment and cloned into the EcoRV-BamHI sitesof a Bluescript M13-SK expression vector containing the humanmethallothionen promoter and the poly A and IVS sequences of the SV40genome (designated pMCR20), to generate plasmid pME1.

To engineer the RSV F gene component of the chimeric construct (FIG. 9,step 2), the RSV F gene lacking the transmembrane region and cytoplasmictail coding regions was retrieved from plasmid pRSVF by cutting thepolylinker with EcoRI and the gene with BspHI. A synthetic BspHI-BamHIoligonucleotide cassette (SEQ ID No: 10) containing three successivetranslational stop codons was ligated to the 1.6 Kb truncated RSV F geneand cloned into the EcoRI-BamHI sites of the Bluescript based expressionvector, pMCR20 to produce plasmid pES13A. Plasmid pES13A then was cutwith EcoRI and PpuMI to remove the leader and F2 coding sequences fromthe truncated RSV F gene. The leader sequence was reconstructed using anEcoRI-PpuMI oligocassette (SEQ ID No: 11) and ligated to the RSV F1 genesegment to generate plasmid pES23A.

To prepare the chimeric F_(PIV-3)-F_(RSV) gene (FIG. 9, step 3)containing the 5′ untranslated region of the PIV-3 F gene linked to thetruncated RSV F1 gene fragment, plasmid pME1 (containing the 1.6 Kbtruncated PIV-3 F gene) first was cut with PpuMI and BamHI. ThePpuMI-BamHI restricted pME1 vector was dephosphorylated with intestinalalkaline phosphatase. The 1.1 Kb RSV F1 gene fragment was retrieved fromplasmid pES23A by cutting the plasmid with PpuMI and BamHI. The 1.1 KbPpuMI-BamHI RSV F1 gene fragment was cloned into the PpuMI-BamHI sitesof the dephosphorylated pME1 vector to generate plasmid pES29A. Thischimeric gene construct contains the 5′ untranslated region of the PIV-3F gene but lacks the nucleotide sequences coding for the hydrophobicanchor domains and cytoplasmic tails of both the PIV-3 and RSV Fproteins.

Example 3

This Example illustrates the construction of a Bluescript-basedexpression vector containing the PIV-3 F gene lacking both the 5′untranslated and transmembrane anchor and cytoplasmic tail codingregions. The steps involved in constructing this plasmid are outlined inFIG. 10.

Plasmid pPI3F containing the full length PIV-3 F gene was cut withBamHI, blunt ended with Klenow polymerase and then cut with BsrI toremove the transmembrane and cytoplasmic tail coding regions. TheBluescript-based expression vector, pMCR20, was cut with SmaI and BamHI.A synthetic BsrI-BamHI oligonucleotide cassette (SEQ ID No: 12)containing a translational stop codon was ligated with the 1. 6 Kb bluntended-BsrI PIV-3 F gene fragment to the SmaI-BamHI restricted pMCR20vector to produce plasmid pMpFB. The PIV-3 F gene of this constructlacked the DNA fragment coding for the transmembrane and cytoplasmicanchor domains but contained the 5′ untranslated region. To engineer aplasmid containing the PIV-3 F gene devoid of both the 5′ untranslatedregion and the DNA fragment coding for the hydrophobic anchor domain,plasmid pMpFB was cut with EcoRI and BstBI. An EcoRI-BstBI oligocassette(SEQ ID No: 13) containing the sequences to reconstruct the signalpeptide and coding sequences removed by the EcoRI-BstBI cut was ligatedto the EcoRI-BstBI restricted pMpFB vector to produce plasmid pMpFA.

Example 4

This Example illustrates the construction of the chimericF_(PIV-3)-F_(RSV) gene composed of the truncated PIV-3 F gene devoid ofthe 5′ untranslated region linked to the truncated RSV F1 gene. Thesteps involved in constructing this plasmid are summarized in FIG. 11.

To prepare this chimeric gene construct, plasmid pES29A (Example 2) wascut with BstBI and BamHI to release the 2.5 Kb BstBI-BamHI PI3-3 F-RSVF1 chimeric gene fragment. This BstBI-BamHI fragment was isolated from alow melting point agarose gel and cloned into the BstBI-BamHI sites ofthe dephosphorylated vector pMpFA to produce plasmid pES60A. Thisconstruct contained the PIV-3 F gene lacking both the 5′ untranslatedregion and the hydrophobic anchor and cytoplasmic tail coding sequenceslinked to the F1 coding region of the truncated RSV F gene. Thischimeric gene was subsequently subcloned into the baculovirus transfervector (see Example 5).

Example 5

This Example illustrates the construction of the modified pAC 610baculovirus transfer vector containing the native polyhedrin promoterand the chimeric F_(PIV-3)-F_(RSV) gene consisting of the PIV-3 F genelacking both the 5′ untranslated sequence and the nucleotide sequencecoding for the hydrophobic anchor domain and cytoplasmic tail linked tothe truncated RSV F1 gene. Construction of this plasmid is illustratedin FIG. 12.

The pAC 610 baculovirus expression vector was modified to contain thenative polyhedrin promoter in the following manner. Vector pAC 610 wascut with EcoRV and BamHI. The 9.4 Kb baculovirus transfer vector lackingthe EcoRV-BamHI DNA sequence was isolated from a low melting pointagarose gel and treated with intestinal alkaline phosphatase. In a 3-wayligation, an EcoRV-EcoRI oligonucleotide cassette (SEQ ID No: 14)containing the nucleotides required to restore the native polyhedrinpromoter was ligated with the 1.6 Kb EcoRI-BamHI truncated RSV F genefragment isolated from construct pES13A (Example 2, step 2) and theEcoRV-BamHI restricted pAC 610 phosphatased vector to generate plasmidpES47A. To prepare the pAC 610 based expression vector containing thechimeric F_(PIV-3)-F_(RSV) gene, plasmid pES47A was first cut with EcoRIand BamHI to remove the 1.6 Kb truncated RSV F gene insert. The 2.8 KbF_(PIV-3)-F_(RSV) chimeric gene was retrieved by cutting plasmid pES60A(Example 4) with EcoRI and BamHI. The 2.8 Kb EcoRI-BamHI chimeric genewas ligated to the EcoRI-BamHI restricted pES47A vector to generateplasmid pAC DR7 (ATCC 75387).

Example 6

This Example outlines the preparation of plaque-purified recombinantbaculoviruses containing the chimeric F_(PIV-3)-F_(RSV) gene.

Spodoptera frugiperda (Sf9) cells were co-transfected with 1.0 μgwild-type AcMNPV DNA and 2.5 μg of F_(PIV-3)-F_(RSV) plasmid DNA(plasmid pAC DR7—Example 5). Putative recombinant baculoviruses(purified once by serial dilution) containing the F_(PIV-3)-F_(RSV)chimeric gene were identified by dot-blot hybridization. Lysates ofinsect cells infected with the putative recombinant baculoviruses wereprobed with the ³²P-labelled FPIV₃-FRSV chimeric gene insert.Recombinant baculoviruses were plaque-purified twice before being usedfor expression studies. All procedures were carried out according to theprotocols outlined by M. D. Summers and G. E. Smith in “A Manual ofMethods for Baculovirus Vectors and Insect Cell Culture Procedures”,Texas Agricultural Experiment Station, Bulletin 1555, 1987.

Example 7

This Example illustrates the presence of the chimeric F_(PIV-3)-F_(RSV)protein in supernatants and cell lysates of infected Sf9 cells.

Insect cells were infected with the plaque-purified recombinantbaculoviruses prepared as described in Example 6 at a m.o.i. of 8.Concentrated supernatants from cells infected with the recombinantviruses were positive in a PIV-3 F specific ELISA. In addition, whenlysates from ³⁵S-methioninelabelled infected cells were subjected toSDS-polyacrylamide gel electrophoresis and gels were analyzed byautoradiography, a strong band with apparent molecular weight ofapproximately 90 kDa was present in lysates of cells infected with therecombinant viruses but was absent in the lysates from wild-typeinfected cells. The presence of the chimeric F_(PIV-3)-F_(RSV) proteinin the lysates of cells infected with the recombinant baculoviruses wasconfirmed further by Western blot analysis using monospecific anti-PIV-3F and anti-RSV F antisera and/or monoclonal antibodies (Mabs). Lysatesfrom cells infected with the recombinant baculoviruses reacted with bothanti-PIV-3 and anti-RSV antisera in immunoblots. As shown in theimmunoblot of FIG. 13, lysates from cells infected with either the RSV For F_(PIV-3)-F_(RSV) recombinant baculoviruses reacted positively withthe anti-F RSV Mab. As expected, lysates from cells infected with wildtype virus did not react with this Mab. In addition, only lysates fromcells infected with the chimeric F_(PIV-3)-F_(RSV) recombinant virusesreacted with the anti-PIV-3 F₁ antiserum.

Example 8

This Example illustrates modification of the baculovirus transfer vectorpVL1392 (obtained from Invitrogen), wherein the polyhedrin ATG startcodon was converted to ATT and the sequence CCG was present downstreamof the polyhedrin gene at positions +4,5,6. Insertion of a structuralgene several base pairs downstream from the ATT codon is known toenhance translation. The steps involved in constructing this modifiedbaculovirus transfer vector are outlined in FIG. 14.

The baculovirus expression vector pVL1392 was cut with EcoRV and BamHI.The 9.5 kb restricted pVL1392 vector was ligated to an EcoRV-BamHIoligonucleotide cassette (SEQ ID No: 15) to produce the pD2 vector.

Example 9

This Example illustrates the construction of the pD2 baculovirusexpression vector containing the chimeric F_(RSV)-HN_(PIV-3) geneconsisting of the truncated RSV F and PIV-3 HN genes linked in tandem.The steps involved in constructing this plasmid are summarized in FIG.15.

To engineer the F_(RSV)-HN_(PIV-3) gene, the RSV F gene lacking thenucleotide sequence coding for the transmembrane domain and cytoplasmictail of the RSV F glycoprotein was retrieved from plasmid pRSVF(Example 1) by cutting the polylinker with EcoRI and the gene withBspHI. The PIV-3 HN gene devoid of the DNA fragment coding for thehydrophobic anchor domain was retrieved from plasmid pPIVHN (Example 1)by cutting the gene with BspHI and the polylinker with BamHI. The 1.6 KbEcoRI-BspHI RSV F gene fragment and the 1.7 Kb BspHI-BamHI PIV-3 HN genefragment were isolated from low melting point agarose gels. For cloningpurposes, the two BspHI sites in the Bluescript based mammalian cellexpression vector, pMCR20, were mutated. Mutations were introduced inthe BspHI sites of the pMCR20 by cutting the expression vector withBspHI, treating both the BspHI restricted vector and the 1. 1 Kbfragment released by the BspHI cut with Klenow polymerase and ligatingthe blunt-ended 1.1 Kb fragment to the blunt-ended Bluescript-basedexpression vector to generate plasmid pM′. Since insertion of the 1.1 Kbblunt-end fragment in the mammalian cell expression vector in theimproper orientation would alter the Ampr gene of the Bluescript-basedexpression vector, only colonies of HB101 cells transformed with the pM′plasmid DNA with the 1.1 Kb blunt-ended fragment in the properorientation could survive in the presence of ampicillin. Plasmid DNA waspurified from ampicillin-resistant colonies of HB101 cells transformedwith plasmid PM′ by equilibrium centrifugation in cesiumchloride-ethidium bromide gradients. The 1.6 Kb EcoRI-BspHI RSV F and1.7 Kb BspHI-BamHI PIV-3 HN gene fragments were directly cloned into theEcoRI-BamHI sites of vector pM′ in a 3-way ligation to generate plasmidpM′ RF-HN.

To restore specific coding sequences of the RSV F and PIV-3 HN genesremoved by the BspHI cut, a BspHI-BspHI oligonucleotide cassette (SEQ IDNo: 16) containing the pertinent RSV F and PIV-3 HN gene sequences wasligated via the BspHI site to the BspHI-restricted plasmid pM′ RF-HN toproduce plasmid pM RF-HN. Clones containing the BspHI-BspHIoligonucleotide cassette in the proper orientation were identified bysequence analysis of the oligonucleotide linker and its flankingregions.

To clone the chimeric F_(RSV)-HN_(PIV-3) gene into the baculovirusexpression vector pD2 (Example 8), the F_(RSV)-HN_(PIV-3) truncated genefirst was retrieved from plasmid pM RF-HN by cutting the plasmid withEcoRI. The 3.3 Kb F_(RSV)-HN_(PIV-3) gene then was cloned into the EcoRIsite of the baculovirus transfer vector plasmid pD2 to generate plasmidpD2 RF-HN (ATCC 75388). Proper orientation of the 3.3 Kb EcoRIF_(RSV)-HN_(PIV-3) chimeric gene insert in plasmid pD2 RF-HN wasconfirmed by sequence analysis.

Example 10

This Example outlines the preparation of plaque-purified recombinantbaculoviruses containing the chimeric F_(RSV)-HN_(PIV-3) gene.

Spodoptera frugiperda (Sf9) cells were co-transfected with 1 μgwild-type AcNPV DNA and 2 μg of F_(RSV)-HN_(PIV-3) plasmid DNA (plasmidpD2 RF-HN-Example 9). Putative recombinant baculoviruses (purified onceby serial dilution) containing the F_(RSV)-HN_(PIV-3) chimeric gene wereidentified by dot-blot hybridization. Lysates of insect cells infectedwith the putative recombinant baculoviruses were probed with the³²P-labelled RSV F or PTV-3 HN gene oligonucleotide probes. Recombinantbaculoviruses were plaque-purified three times before being used forexpression studies. All procedures were carried out according to theprotocols outlined by Summers and Smith (Example 6).

Example 11

This Example illustrates the presence of the chimeric F_(RSV)-HN_(PIV-3)protein in supernatants of infected Sf9 and High 5 cells.

Insect cells (Sf9 and High 5), maintained in serum free medium EX401,were infected with the plaque purified recombinant baculoviruses ofExample 10 at a m.o.i. of 5 to 10 pfu/cell. Supernatants from cellsinfected with the recombinant baculoviruses tested positive forexpressed protein in both the RSV-F and PIV-3 HN specific ELISAS. Inaddition, supernatants from infected cells reacted positively with bothan anti-F RSV monoclonal antibody and anti-HN peptide antisera onimmunoblots. A distinct band of approximately 105 kDa was present in theimmunoblots. These results confirm the secretion of the chimericF_(RSV)-HN_(PIV-3) protein into the supernatant of Sf9 and High 5 cellsinfected with the recombinant baculoviruses.

Example 12

This Example illustrates the purification of the chimericF_(RSV)-HN_(PIV-3) protein from the supernatants of infected High 5cells.

High 5 cells, maintained in serum free medium, were infected with theplaque purified recombinant baculoviruses of Example 10 at a m.o.i of 5pfu/cell. The supernatant from virus infected cells was harvested 2 dayspost-infection C he soluble F_(RSV)-HN_(PIV-3) chimeric protein waspurified the supernatants of infected cells by immunoaffinitychromatography using an anti-HN PIV-3 monoclonal antibody. The anti-HNmonoclonal antibody was coupled to CNBr-activated Sepharose 4B byconventional techniques. The immunoaffinity column was washed with 10bed volumes of washing buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.02%v/v TRITON-X 100 (Trademark for a non-ionic detergent)) prior to use.After sample loading, the column was washed with 10 bed volumes ofwashing buffer followed by 3 bed volumes of high salt buffer (10 mmTris-HCl pH 7.5, 500 mM NaCl, 0.02% v/v Triton-X 100). The chimericF_(RSV)-HN_(PIV-3) protein was eluted from the immunoaffinity columnwith 100 MM glycine, pH 2.5, in the presence of 0.02% TRITON X-100.Eluted protein was neutralized immediately with 1M Tris-HCl, pH 10.7.

Polyacrylamide gel electrophoretic analysis (FIG. 16, panel A) of theimmunoaffinity-purified F_(RSV)-HN_(PIV-3) protein revealed the presenceof one major protein band with an apparent molecular weight of 105 kDa.The purified protein reacted with both an anti-RSV F monoclonal antibodyand anti-HN peptide antisera on immunoblots (FIG. 16, panel B, lanes 1and 2, respectively).

Example 13

This Example illustrates the immunogenicity of the F_(RSV)-HN_(PIV-3)protein in guinea pigs.

Groups of four guinea pigs were injected intramuscularly with either 1.0or 10.0 μg of the chimeric F_(RSV)-HN_(PIV-3) protein purified asdescribed in Example 12 and adjuvanted with aluminum phosphate. Groupsof control animals were immunized with either placebo, or live PIV-3 orRSV (administered intranasally). Guinea pigs were bled 2 and 4 weeksafter the primary injection and boosted at 4 weeks with an equivalentdose of the antigen formulation. Serum samples also were taken 2 and 4weeks after the booster dose. To assess-the ability of the chimericprotein to elicit PIV-3 and RSV-specific antibody responses, serasamples were analyzed for the presence of PIV-3 specifichemagglutination inhibiting and neutralizing antibodies as well as RSVneutralizing antibodies. As summarized in Table 1 below (the Tablesappear at the end of the disclosure), the sera of animals immunized withtwo 10 μg doses of the chimeric protein had titres of PIV-3 specifichemagglutination inhibition (HAI) and PIV-3/RSV neutralizing antibodiesat the 6 and 8 week time points which were equivalent to the levelsobtained following intranasal inoculation with either live PIV-3 or RSV.In addition, animals immunized with only two 1 ug doses of the chimericprotein elicited strong PIV-3 and RSV specific neutralizing antibodies.These results confirmed the immunogenicity of both the RSV and PIV-3components of the chimeric protein and provided confirmatory evidencethat a single recombinant immunogen can elicit neutralizing antibodiesagainst both RSV and PIV-3.

Example 14

This Example illustrates the immunogenicity and protective ability ofthe F_(RSV)-HN_(PIV-3) protein in cotton rats.

Groups of eight cotton rats were injected intramuscularly with either1.0 or 10.0 ug of the chimeric F_(RSV)-HN_(PIV-3) protein (prepared asdescribed in Example 12) adjuvanted with aluminum phosphate. Groups ofcontrol animals were immunized with either placebo (PBS+aluminumphosphate) or live PIV-3 or RSV (administered intranasally). Cotton ratswere bled 4 weeks after the primary injection and boosted at 4 weekswith an equivalent dose of the antigen formulation. Serum samples werealso taken 1 week after the booster dose. As shown in Table 2 below,data from the 4-week bleed demonstrated that both a 1 and 10 μg dose ofthe chimeric protein was capable of inducing a strong primary response.Reciprocal mean log₂ PIV-3 specific HAI and PIV-3/RSV neutralizingtiters were equivalent to the titres obtained with live PIV-3 and RSV.Thus, a single inoculation of the chimeric protein was sufficient toelicit neutralizing antibodies against both PIV-3 and RSV. Strongneutralizing PIV-3 and RSV titres also were observed following thebooster dose (5 week bleed). These results provide additional evidencethat both the RSV and PIV-3 components of the chimeric protein arehighly immunogenic.

To assess the ability of the chimeric immunogen to simultaneouslyprotect animals against both RSV and PIV-3, four cotton rats from eachgroup were challenged intranasally with 100 TCID₅₀ units of either PIV-3or RSV. Animals were killed 4 days after virus challenge. Virus titerswere determined in lung lavages. As shown in Table 3 below, animalsimmunized with either 1 or 10 μg of the chimeric F_(RSV)-HN_(PIV-3)protein were completely protected against challenge with either PIV-3 orRSV. These results provide evidence that the chimeric protein is notonly highly immunogenic but can also simultaneously protect cotton ratsagainst disease caused by both PIV-3 and RSV infection.

Example 15

This Example illustrates the construction of a Bluescript M13-SK vectorcontaining the chimeric F_(PIV-3)-G_(RSV) gene. This chimeric geneconstruct contains the 5′ untranslated region of a mutated PIV-3 F genebut lacks the nucleotide sequence coding for the hydrophobic anchor andcytoplasmic tail domains of both a mutated PIV-3 F and the native RSV Ggenes. The steps involved in constructing this plasmid are outlined inFIGS. 17 and 18.

The first step (FIG. 17) involved in preparing the PIV-3 F component ofthe chimeric F_(PIV-3)-G_(RSV) gene construct was to eliminate theputative pre-termination sites within the 18 nucleotide long sequence 5′CAAGAAAAAGGAATAAAA 3′ (SEQ ID No: 17) located between positions 857 and874 of the non PCR-amplified PIV-3 F gene and positions 847 and 864 ofthe PCR-amplified PIV-3 F gene (see FIG. 1). To this end, the PIV-F cDNAof the non-PCR amplified PIV-3 F gene was cut at the BsaAI and EcoRIsites. The BsaAI-EcoRI PIV F gene fragment was cloned into the EcoRIsite of a Bluescript M13-SK vector using an EcoRI-BsaAI linker. The857-874 target region of the PIV-3 F gene (non-PCR amplified) then wasmutated by oligonucleotide-mediated mutagenesis using the method ofMorinaga et al. [1984, Biotechnology 2: 636-639]. Plasmid pPI3Fc(Example 1) was cut with ScaI in the Amp^(r) gene and dephosphorylatedwith alkaline phosphatase (plasmid #1). A second sample of plasmidpPI3Fc was cut with BstEII and NsiI to produce a 3.9 Kb restrictedplasmid, lacking the 0.9 Kb BstEII-NsiI fragment of the PIV-3 F gene(plasmid #2). A mutagenic 78-mer synthetic oligonucleotide (#2721 shownin FIG. 17-SEQ ID No: 18)) containing the sequence 5′ CAGGAGAAGGGTATCAAG3′ (SEQ ID No: 19) was synthesized to specifically mutate the 857-874DNA segment without changing the F protein sequence. Thisoligonucleotide was added to plasmid DNAs #1 and #2, denatured at 100°C. for 3 min. and renatured by gradual cooling. The mixture then wasincubated in the presence of DNA polymerase, dNTPs and T4 ligase andtransformed into HB101 cells. Bacteria containing the 1.8 Kb mutatedPIV-3 F gene were isolated on YT agar plates containing 100 μg/mlampicillin. Hybridization with the oligonucleotide probe 5′AGGAGAAGGGTATCAAG 3′ (SEQ ID No: 20) was used to confirm the presence ofthe mutated PIV-3 F gene. The mutated gene sequence was confirmed by DNAsequencing. The plasmid containing the mutated PIV-3 gene was designatedpPI3Fm.

The second step (FIG. 18) in the engineering of the chimeric geneconstruct involved constructing a Bluescript based vector to contain thetruncated PIV-3 Fm gene lacking the nucleotide sequence coding for thetransmembrane anchor domain and cytoplasmic tail of the PIV-3 F proteinlinked in tandem with the RSV G gene lacking both the 5′ leader sequenceand the nucleotide sequence coding for the transmembrane anchor domainand cytoplasmic tail of the G glycoprotein.

To prepare this chimeric gene, the orientation of the mutated PIV-F genein plasmid pPI3Fm first was reversed by EcoRI digestion and religationto generate plasmid pPI3Fmr. To prepare the PIV-3 F gene component ofthe chimeric gene, plasmid pPI3Fmr was cut with NotI and BsrI to releasethe 1.7 Kb truncated PIV-3 F gene. To prepare the RSV G component, the0.95 Kb RSV-G gene lacking both the 5′ leader sequence and the DNAsegment encoding the G protein anchor domain and cytoplasmic tail wasreleased from plasmid PRSVG (Example 1) by cutting the polylinker withEcoRI and the gene with BamHI. The 0.95 Kb EcoRI-BamHI RSV G genefragment was subcloned into the EcoRI-BamHI sites of a restrictedBluescript vector, pMl3-SK, to produce plasmid pRSVGt. The 0.95 KbEcoRI-BamHI G gene fragment and the 1.5 Kb NotI-BsrI truncated PIV-3 Fgene were linked via a BsrI-BamHI oligonucleotide cassette (SEQ ID No:9) restoring the F and G gene coding sequences and cloned into thepRSVGt vector restricted with BamHI and NotI in a 3-way ligation. Theplasmid thus generated was designated pFG.

Example 16

This Example outlines the construction of the pD2 baculovirus transfervector (described in Example 8) containing the chimericF_(PIV-3)-G_(RSV) gene consisting of a mutated PIV-3 F gene lacking thehydrophobic anchor and cytoplasmic coding regions linked to the RSV Ggene lacking both the 5′ leader sequence and the nucleotide sequencesencoding the transmembrane anchor domain and cytoplasmic tail of the Gprotein.

To prepare this construct, plasmid pFG (Example 15) was cut with EcoRIto release the 2.6 Kb F_(PIV-3)-G_(RSV) chimeric gene. The 2.6 Kb EcoRIrestricted chimeric gene fragment then was sub-cloned into the EcoRIsite of the dephosphorylated pD2 vector to generate the 12.1 Kb plasmidpD2F-G (ATCC 75389).

Example 17

This Example outlines the preparation of plaque-purified recombinantbaculoviruses containing the chimeric F_(PIV-3)-G_(RSV) gene.

Spodoptera frugiperda (Sf9) cells were co-transfected with 2 ug ofpD2F-G plasmid DNA (Example 16) and 1 ug of linear wild-type AcNPV DNA(obtained from Invitrogen). Recombinant baculoviruses containing theF_(PIV-3)-G_(RSV) gene were plaque-purified twice according to theprocedure outlined in Example 10.

Example 18

This Example illustrates the presence of the chimeric F_(PIV-3)-G_(RSV)protein in the supernatant of Sf9 and High 5 cells infected with therecombinant baculoviruses.

Sf9 and High 5 cells were infected with recombinant baculovirusescontaining the F_(PIV-3)-G_(RSV) gene (Example 16) at a m.o.i. of 5 to10 pfu/cell. The supernatant of cells infected with the recombinantviruses tested positive for expressed protein in the PIV-3 F specificELISA. Supernatants of infected cells reacted with both anti-F PIV-3 andanti-G RSV monoclonal antibodies in immunoblots. These results confirmthe presence of the chimeric F_(PIV-3)-G_(RSV) protein in thesupernatants of infected Sf9 and High 5 cells.

Example 19

This Example outlines the preparation of recombinant vaccinia virusesexpressing the F_(PIV-3)-F_(RSV) and F_(RSV)-HN_(PIV-3) genes.

Vaccinia virus recombinant viruses expressing the F_(PIV-3)-F_(RSV)(designated vP1192) and F_(RSV)-HN_(PIV-3) (designated vP1195) geneswere produced at Virogenetics Corporation (Troy, N.Y.) (an entityrelated to assignee hereof) using the COPAK host-range selection system.Insertion plasmids used in the COPAK host-range selection systemcontained the vaccinia K1L host-range gene [Perkus et al., (1990)Virology 179:276-286] and the modified vaccinia H6 promoter [Perkus etal. (1989), J. Virology 63:3829-3836]. In these insertion plasmids, theK1L gene, H6 promoter and polylinker region are situated betweenCopenhagen strain vaccinia flanking arms replacing the ATI region [openreading frames (ORFS) A25L, A26L; Goebel et al., (1990), Virology 179:247-266; 517-563]. COPAK insertion plasmids are designed for use in invivo recombination using the rescue virus NYVAC (vP866) (Tartaglia etal., (1992) Virology 188: 217-232). Selection of recombinant viruses wasdone on rabbit kidney cells.

Recombinant viruses, vP1192 and vP1195 were generated using insertionplasmids pES229A-6 and PSD.RN, respectively. To prepare plasmidpES229A-6 containing the F_(PIV-3)-F_(RSV) gene, the COPAK-H6 insertionplasmid pSD555 was cut with SmaI and dephosphorylated with intestinalalkaline phosphatase. The 2.6 Kb F_(PIV-3)-F_(RSV) gene was retrievedfrom plasmid pES60A (Example 4) by cutting the plasmid with EcoRI andBamHI. The 2.6 Kb EcoRI-BamHI F_(PIV-3)-F_(RSV) gene was blunt endedwith Klenow polymerase, isolated from a low melting point agarose geland cloned into the SmaI site of the COPAK-H6 insertion plasmid pSD555to generate plasmid pES229A-6. This positioned the F_(PIV-3)-F_(RSV) ORFsuch that the 5′ end is nearest the H6 promoter.

To prepare plasmid PSD.RN, the pSD555 vector first was cut with SmaI andBamHI. Plasmid pM RF-HN (Example 9) containing the truncatedF_(RSV)-HN_(PIV-3) gene was cut with ClaI, blunt ended with Klenowpolymerase and then cut with BamHI. The 3.3 Kb F_(RSV)-HN_(PIV-3) genewas cloned into the SmaI-BamHI sites of the pSD555 vector to generateplasmid PSD.RN. This positioned the F_(RSV)-HN_(PIV-3) ORF such that theH6 5′ end is nearest the H6 promoter.

Plasmids pES229A-6 and PSD.RN were used in in vitro recombinationexperiments in vero cells with NYVAC (vP866) as the rescuing virus.Recombinant progeny virus was selected on rabbit kidney (RK)-13 cells(ATCC #CCL37). Several plaques were passaged two times on RK-13 cells.Virus containing the chimeric genes were confirmed by standard in situplaque hybridization [Piccini et al. (1987), Methods in Enzymology,153:545-563] using radiolabeled probes specific for the PIV and RSVinserted DNA sequences. Plaque purified virus containing theF_(PIV-3)-F_(RSV) and F_(RSV)-HN_(PIV-3) chimeric genes were designatedvP1192 and vP1195, respectively.

Radioimmunoprecipitation was done to confirm the expression of thechimeric genes in vP1192 and vP1195 infected cells. These assays wereperformed with lysates prepared from infected Vero cells [according tothe procedure of Taylor et al., (1990) J. Virology 64, 1441-1450] usingguinea pig monospecific PIV-3 anti-HN and anti-F antiserum and rabbitanti-RSV F antiserum. Both the anti-PIV F and anti-RSV F antiseraprecipitated a protein with an apparent molecular weight ofapproximately 90 koa from vP1192 infected Vero cells. Both anti-RSV Fand guinea pig anti-PIV HN antisera precipitated a protein with anapparent molecular weight of approximately 100 kDa from vP1195 infectedcells. These results confirmed the production of the F_(PIV-3)-F_(RSV)and F_(RSV)-HN_(PIV-3) chimeric proteins in Vero cells infected with therecombinant poxviruses.

Summary of Disclosure

In summary of the disclosure, the present invention provides multimerichybrid genes which produce chimeric proteins capable of elicitingprotection against infection by a plurality of pathogens, particularlyPIV and RSV. Modifications are possible within the scope of thisinvention.

TABLE 1 Secondary antibody response of guinea pigs immunized with thechimeric F_(RSV)-HN_(PIV-3) protein HAI Titre^(a) NeutratizationTitre^(b) (log₂ ± s.e.) (log₂ ± s.e.) Antigen Dose PIV-3 PIV-3 RSVFormulation (ug) 6 wk Bleed 8 wk Bleed 6 wk Bleed 8 wk Bleed 6 wk Bleed8 wk Bleed Buffer — <1.0 ± 0.0  <1.0 ± 0.0  <1.0 ± 0.0  <1.0 ± 0.0  <1.0± 0.0  <1.0 ± 0.0  F_(RSV)-HN_(PIV-3) 10.0 9.1 ± 0.3 9.1 ± 0.3 7.1 ± 0.37.1 ± 0.5 5.5 ± 0.9 4.5 ± 1.2  1.0 7.0 ± 2.0 7.3 ± 2.2 5.0 ± 1.5 4.5 ±1.4 4.5 ± 0.5 3.0 ± 1.0 Live PIV-3 8.6 ± 0.7 7.3 ± 0.6 7.0 ± 0.4 7.3 ±0.6 N/A N/A Live RSV N/A^(c) N/A N/A N/A 5.5 ± 1.5 5.0 ± 1.0^(a)Reciprocal mean log₂ serum dilution which inhibits erythrocyteagglutination by 4 hemagglutinating units of PIV-3 ^(b)Reciprocal meanlog₂ serum dilution which blocks hemadsorption of 100 TCID₅₀ units ofPIV-3 or RSV ^(c)N/A—not applicable

TABLE 2 Serum antibody response of cotton rats immunized with thechimeric F_(RSV)-HN_(PIV-3) protein^(a) HAI Titre^(b) NeutratizationTitre^(c) (log₂ ± s.e.) (log₂ ± s.e.) Antigen Dose PIV-3 PIV-3 RSVFormulation (ug) 4 wk Bleed 5 wk Bleed 4 wk Bleed 5 wk Bleed 4 wk Bleed5 wk Bleed Buffer — 2.8 ± 0.5 <3.0 ± 0.0 <1.0 ± 1.0 <1.0 ± 0.0 1.8 ± 0.30.8 ± 0.7 F_(RSV)-HN_(PIV-3) 10.0 9.5 ± 1.3 10.5 ± 0.6 >9.0 ± 0.0 >9.0 ±0.0 5.2 ± 1.1 5.8 ± 0.9  1.0 9.3 ± 1.0 10.3 ± 0.5 >9.0 ± 0.0 >9.0 ± 0.05.0 ± 0.7 5.8 ± 1.2 Live PIV-3 7.0 ± 0.0  8.5 ± 0.7 >9.0 ± 0.0  9.2 ±0.7 N/A N/A Live RSV N/A^(d) N/A N/A N/A 5.5 ± 0.6 8.5 ± 0.6 ^(a)Eachvalue represents the mean titre of antisera from 8 animals.^(b)Reciprocal mean log₂ serum dilution which inhibits erythrocyteagglutination by 4 hemagglutinating units of PIV-3 ^(c)Reciprocal meanlog₂ serum dilution which blocks hemadsorption of 100 TCID₅₀ units ofPIV-3 or RSV ^(d)N/A—not applicable

TABLE 3 Response of immunized cotton rats to PIV/RSV challenge^(a) Meanvirus lung titre Antigen Dose log ₁₀/g lung ± s.d. Formulation (ug) RSVPIV-3 Buffer —  3.7 ± 0.3  3.4 ± 0.3 F_(RSV)-HN_(PIV-3) 10.0 ≦1.5 ± 0.0≦1.5 ± 0.0 F_(RSV)-HN_(PIV-3)  1.0 ≦1.5 ± 0.0 ≦1.5 ± 0.0 Live RSV ≦1.5 ±0.0 ≦1.5 ± 0.0 Live PIV-3 ≦1.5 ± 0.0 ≦1.5 ± 0.0 ^(a)Animals werechallenged intranasally with 100 TCID₅₀ units of PIV-3 or RSV and killed4 days later. Each value represents the mean virus lung titre of 4animals.

21 1844 base pairs nucleic acid single linear DNA (genomic) unknown 1AAGTCAATAC CAACAACTAT TAGCAGTCAT ACGTGCAAGA ACAAGAAAGA AGAGATTCAA 60AAAGCTAAAT AAGAGAAATC AAAACAAAAG GTATAGAACA CCCGAACAAC AAAATCAAAA 120CATCCAATCC ATTTTAAACA AAAATTCCAA AAGAGACCGG CAACACAACA AGCACCAAAC 180ACAATGCCAA CTTTAATACT GCTAATTATT ACAACAATGA TTATGGCATC TTCCTGCCAA 240ATAGATATCA CAAAACTACA GCATGTAGGT GTATTGGTCA ACAGTCCCAA AGGGATGAAG 300ATATCACAAA ACTTCGAAAC AAGATATCTA ATTTTGAGCC TCATACCAAA AATAGAAGAC 360TCTAACTCTT GTGGTGACCA ACAGATCAAA CAATACAAGA GGTTATTGGA TAGACTGATC 420ATCCCTCTAT ATGATGGATT AAGATTACAG AAAGATGTGA TAGTAACCAA TCAAGAATCC 480AATGAAAACA CTGATCCCAG AACAAGACGA TCCTTTGGAG GGGTAATTGG AACCATTGCT 540CTGGGAGTAG CAACCTCAGC ACAAATTACA GCGGCAGTTG CTCTGGTTGA AGCCAAGCAG 600GCAAAATCAG ACATCGAAAA ACTCAAAGAA GCAATCAGGG ACACAAACAA AGCAGTGCAG 660TCAGTTCAGA GCTCTATAGG AAATTTAATA GTAGCAATTA AATCAGTCCA AGATTATGTC 720AACAACGAAA TGGTGCCATC GATTGCTAGA CTAGGTTGTG AAGCAGCAGG ACTTCAATTA 780GGAATTGCAT TAACACAGCA TTACTCAGAA TTAACAAACA TATTTGGTGA TAACATAGGA 840TCGTTACAAG AAAAAGGAAT AAAATTACAA GGTATAGCAT CATTATACCG CACAAATATC 900ACAGAAATAT TCACAACATC AACAGTTGAT AAATATGATA TCTATGATCT ATTATTTACA 960GAATCAATAA AGGTGAGAGT TATAGATGTT GATTTGAATG ATTACTCAAT CACCCTCCAA 1020GTCAGACTCC CTTTATTAAC TAGGCTGCTG AACACTCAGA TCTACAAAGT AGATTCCATA 1080TCATATAATA TCCAAAACAG AGAATGGTAT ATCCCTCTTC CCAGCCATAT CATGACGAAA 1140GGGGCATTTC TAGGTGGAGC AGATGTCAAG GAATGTATAG AAGCATTCAG CAGTTATATA 1200TGCCCTTCTG ATCCAGGATT TGTACTAAAC CATGAAATGG AGAGCTGCTT ATCAGGAAAC 1260ATATCCCAAT GTCCAAGAAC CACGGTCACA TCAGACATTG TTCCAAGATA TGCATTTGTC 1320AATGGAGGAG TGGTTGCAAA CTGTATAACA ACCACCTGTA CATGCAACGG AATCGACAAT 1380AGAATCAATC AACCACCTGA TCAAGGAGTA AAAATTATAA CACATAAAGA ATGTAATACA 1440ATAGGTATCA ACGGAATGCT GTTCAATACA AATAAAGAAG GAACTCTTGC ATTCTACACA 1500CCAAATGATA TAACACTAAA TAATTCTGTT GCACTTGATC CAATTGACAT ATCAATCGAG 1560CTTAACAAAG CCAAATCAGA TCTAGAAGAA TCAAAAGAAT GGATAAGAAG GTCAAATCAA 1620AAACTAGATT CTATTGGAAA CTGGCATCAA TCTAGCACTA CAATCATAAT TATTTTAATA 1680ATGATCATTA TATTGTTTAT AATTAATGTA ACGATAATTA CAATTGCAAT TAAGTATTAC 1740AGAATTCAAA AGAGAAATCG AGTGGATCAA AATGACAAGC CATATGTACT AACAAACAAA 1800TGACATATCT ATAGATCATT AGATATTAAA ATTATAAAAA ACTT 1844 539 amino acidsamino acid single linear DNA (genomic) unknown 2 Met Pro Thr Leu Ile LeuLeu Ile Ile Thr Thr Met Ile Met Ala Ser 1 5 10 15 Ser Cys Gln Ile AspIle Thr Lys Leu Gln His Val Gly Val Leu Val 20 25 30 Asn Ser Pro Lys GlyMet Lys Ile Ser Gln Asn Phe Glu Thr Arg Tyr 35 40 45 Leu Ile Leu Ser LeuIle Pro Lys Ile Glu Asp Ser Asn Ser Cys Gly 50 55 60 Asp Gln Gln Ile LysGln Tyr Lys Arg Leu Leu Asp Arg Leu Ile Ile 65 70 75 80 Pro Leu Tyr AspGly Leu Arg Leu Gln Lys Asp Val Ile Val Thr Asn 85 90 95 Gln Glu Ser AsnGlu Asn Thr Asp Pro Arg Thr Arg Arg Ser Phe Gly 100 105 110 Gly Val IleGly Thr Ile Ala Leu Gly Val Ala Thr Ser Ala Gln Ile 115 120 125 Thr AlaAla Val Ala Leu Val Glu Ala Lys Gln Ala Lys Ser Asp Ile 130 135 140 GluLys Leu Lys Glu Ala Ile Arg Asp Thr Asn Lys Ala Val Gln Ser 145 150 155160 Val Gln Ser Ser Ile Gly Asn Leu Ile Val Ala Ile Lys Ser Val Gln 165170 175 Asp Tyr Val Asn Asn Glu Ile Val Pro Ser Ile Ala Arg Leu Gly Cys180 185 190 Glu Ala Ala Gly Leu Gln Leu Gly Ile Ala Leu Thr Gln His TyrSer 195 200 205 Glu Leu Thr Asn Ile Phe Gly Asp Asn Ile Gly Ser Leu GlnGlu Lys 210 215 220 Gly Ile Lys Leu Gln Gly Ile Ala Ser Leu Tyr Arg ThrAsn Ile Thr 225 230 235 240 Glu Ile Phe Thr Thr Ser Thr Val Asp Lys TyrAsp Ile Tyr Asp Leu 245 250 255 Leu Phe Thr Glu Ser Ile Lys Val Arg ValIle Asp Val Asp Leu Asn 260 265 270 Asp Tyr Ser Ile Thr Leu Gln Val ArgLeu Pro Leu Leu Thr Arg Leu 275 280 285 Leu Asn Thr Gln Ile Tyr Lys ValAsp Ser Ile Ser Tyr Asn Ile Gln 290 295 300 Asn Arg Glu Trp Tyr Ile ProLeu Pro Ser His Ile Met Thr Lys Gly 305 310 315 320 Ala Phe Leu Gly GlyAla Asp Val Lys Glu Cys Ile Glu Ala Phe Ser 325 330 335 Ser Tyr Ile CysPro Ser Asp Pro Gly Phe Val Leu Asn His Glu Met 340 345 350 Glu Ser CysLeu Ser Gly Asn Ile Ser Gln Cys Pro Arg Thr Thr Val 355 360 365 Thr SerAsp Ile Val Pro Arg Tyr Ala Phe Val Asn Gly Gly Val Val 370 375 380 AlaAsn Cys Ile Thr Thr Thr Cys Thr Cys Asn Gly Ile Asp Asn Arg 385 390 395400 Ile Asn Gln Pro Pro Asp Gln Gly Val Lys Ile Ile Thr His Lys Glu 405410 415 Cys Asn Thr Ile Gly Ile Asn Gly Met Leu Phe Asn Thr Asn Lys Glu420 425 430 Gly Thr Leu Ala Phe Tyr Thr Pro Asn Asp Ile Thr Leu Asn AsnSer 435 440 445 Val Ala Leu Asp Pro Ile Asp Ile Ser Ile Glu Leu Asn LysAla Lys 450 455 460 Ser Asp Leu Glu Glu Ser Lys Glu Trp Ile Arg Arg SerAsn Gln Lys 465 470 475 480 Leu Asp Ser Ile Gly Asn Trp His Gln Ser SerThr Thr Ile Ile Ile 485 490 495 Ile Leu Ile Met Ile Ile Ile Leu Phe IleIle Asn Val Thr Ile Ile 500 505 510 Thr Ile Ala Ile Lys Tyr Tyr Arg IleGln Lys Arg Asn Arg Val Asp 515 520 525 Gln Asn Asp Lys Pro Tyr Val LeuThr Asn Lys 530 535 1833 base pairs nucleic acid single linear DNA(genomic) unknown 3 AGACAAATCC AAATTCGAGA TGGAATACTG GAAGCATACCAATCACGGAA AGGATGCTGG 60 CAATGAGCTG GAGACGTCCA TGGCTACTAA TGGCAACAAGCTCACCAATA AGATAACATA 120 TATATTATGG ACAATAATCC TGGTGTTATT ATCAATAGTCTTCATCATAG TGCTAATTAA 180 TTCCATCAAA AGTGAAAAGG CTCATGAATC ATTGCTGCAAGACATAAATA ATGAGTTTAT 240 GGAAATTACA GAAAAGATCC AAATGGCATC GGATAATACCAATGATCTAA TACAGTCAGG 300 AGTGAATACA AGGCTTCTTA CAATTCAGAG TCATGTCCAGAATTATATAC CAATATCACT 360 GACACAACAG ATGTCAGATC TTAGGAAATT CATTAGTGAAATTACAATTA GAAATGATAA 420 TCAAGAAGTG CTGCCACAAA GAATAACACA TGATGTGGGTATAAAACCTT TAAATCCAGA 480 TGATTTTTGG AGATGCACGT CTGGTCTTCC ATCTTTAATGAAAACTCCAA AAATAAGGTT 540 AATGCCAGGG CCGGGATTAT TAGCTATGCC AACGACTGTTGATGGCTGTA TCAGAACTCC 600 GTCCTTAGTT ATAAATGATC TGATTTATGC TTATACCTCAAATCTAATTA CTCGAGGTTG 660 TCAGGATATA GGAAAATCAT ATCAAGTCTT ACAGATAGGGATAATAACTG TAAACTCAGA 720 CTTGGTACCT GACTTAAATC CCAGGATCTC TCATACTTTTAACATAAATG ACAATAGGAA 780 GTCATGTTCT CTAGCACTCC TAAATACAGA TGTATATCAACTGTGTTCAA CTCCCAAAGT 840 TGATGAAAGA TCAGATTATG CATCATCAGG CATAGAAGATATTGTACTTG ATATTGTCAA 900 TTATGATGGC TCAATCTCAA CAACAAGATT TAAGAATAATAACATAAGCT TTGATCAACC 960 TTATGCTGCA CTATACCCAT CTGTTGGACC AGGGATATACTACAAAGGCA AAATAATATT 1020 TCTCGGGTAT GGAGGTCTTG AACATCCAAT AAATGAGAATGTAATCTGCA ACACAACTGG 1080 GTGTCCCGGG AAAACACAGA GAGACTGCAA TCAGGCATCTCATAGTCCAT GGTTTTCAGA 1140 TAGGAGGATG GTCAACTCTA TCATTGTTGT TGACAAAGGCTTAAACTCAA TTCCAAAATT 1200 GAAGGTATGG ACGATATCTA TGAGACAGAA TTACTGGGGGTCAGAAGGAA GGTTACTTCT 1260 ACTAGGTAAC AAGATCTATA TATATACAAG ATCCACAAGTTGGCATAGCA AGTTACAATT 1320 AGGAATAATT GATATTACTG ATTACAGTGA TATAAGGATAAAATGGACAT GGCATAATGT 1380 GCTATCAAGA CCAGGAAACA ATGAATGTCC ATGGGGACATTCATGTCCAG ATGGATGTAT 1440 AACAGGAGTA TATACTGATG CATATCCACT CAATCCCACAGGGAGCATTG TGTCATCTGT 1500 CATATTAGAT TCACAAAAAT CGAGAGTGAA CCCAGTCATAACTTACTCAA CAGCAACCGA 1560 AAGAGTAAAC GAGCTGGCCA TCCGAAACAG AACACTCTCAGCTGGATATA CAACAACAAG 1620 CTGCATCACA CACTATAACA AAGGATATTG TTTTCATATAGTAGAAATAA ATCAGAAAAG 1680 CTTAAACACA CTTCAACCCA TGTTGTTCAA GACAGAGGTTCCAAAAAGCT GCAGTTAATC 1740 ATAATTAACC GCAATATGCA TTAACCTATC TATAATACAAGTATATGATA AGTAATCAGC 1800 AATCAGACAA TAGACAAAAG GGAAATATAA AAA 1833 572amino acids amino acid single linear DNA (genomic) unknown 4 Met Glu TyrTrp Lys His Thr Asn His Gly Lys Asp Ala Gly Asn Glu 1 5 10 15 Leu GluThr Ser Met Ala Thr Asn Gly Asn Lys Leu Thr Asn Lys Ile 20 25 30 Thr TyrIle Leu Trp Thr Ile Ile Leu Val Leu Leu Ser Ile Val Phe 35 40 45 Ile IleVal Leu Ile Asn Ser Ile Lys Ser Glu Lys Ala His Glu Ser 50 55 60 Leu LeuGln Asp Ile Asn Asn Glu Phe Met Glu Ile Thr Glu Lys Ile 65 70 75 80 GlnMet Ala Ser Asp Asn Thr Asn Asp Leu Ile Gln Ser Gly Val Asn 85 90 95 ThrArg Leu Leu Thr Ile Gln Ser His Val Gln Asn Tyr Ile Pro Ile 100 105 110Ser Leu Thr Gln Gln Met Ser Asp Leu Arg Lys Phe Ile Ser Glu Ile 115 120125 Thr Ile Arg Asn Asp Asn Gln Glu Val Leu Pro Gln Arg Ile Thr His 130135 140 Asp Val Gly Ile Lys Pro Leu Asn Pro Asp Asp Phe Trp Arg Cys Thr145 150 155 160 Ser Gly Leu Pro Ser Leu Met Lys Thr Pro Lys Ile Arg LeuMet Pro 165 170 175 Gly Pro Gly Leu Leu Ala Met Pro Thr Thr Val Asp GlyCys Ile Arg 180 185 190 Thr Pro Ser Leu Val Ile Asn Asp Leu Ile Tyr AlaTyr Thr Ser Asn 195 200 205 Leu Ile Thr Arg Gly Cys Gln Asp Ile Gly LysSer Tyr Gln Val Leu 210 215 220 Gln Ile Gly Ile Ile Thr Val Asn Ser AspLeu Val Pro Asp Leu Asn 225 230 235 240 Pro Arg Ile Ser His Thr Phe AsnIle Asn Asp Asn Arg Lys Ser Cys 245 250 255 Ser Leu Ala Leu Leu Asn ThrAsp Val Tyr Gln Leu Cys Ser Thr Pro 260 265 270 Lys Val Asp Glu Arg SerAsp Tyr Ala Ser Ser Gly Ile Glu Asp Ile 275 280 285 Val Leu Asp Ile ValAsn Tyr Asp Gly Ser Ile Ser Thr Thr Arg Phe 290 295 300 Lys Asn Asn AsnIle Ser Phe Asp Gln Pro Tyr Ala Ala Leu Tyr Pro 305 310 315 320 Ser ValGly Pro Gly Ile Tyr Tyr Lys Gly Lys Ile Ile Phe Leu Gly 325 330 335 TyrGly Gly Leu Glu His Pro Ile Asn Glu Asn Val Ile Cys Asn Thr 340 345 350Thr Gly Cys Pro Gly Lys Thr Gln Arg Asp Cys Asn Gln Ala Ser His 355 360365 Ser Pro Trp Phe Ser Asp Arg Arg Met Val Asn Ser Ile Ile Val Val 370375 380 Asp Lys Gly Leu Asn Ser Ile Pro Lys Leu Lys Val Trp Thr Ile Ser385 390 395 400 Met Arg Gln Asn Tyr Trp Gly Ser Glu Gly Arg Leu Leu LeuLeu Gly 405 410 415 Asn Lys Ile Tyr Ile Tyr Thr Arg Ser Thr Ser Trp HisSer Lys Leu 420 425 430 Gln Leu Gly Ile Ile Asp Ile Thr Asp Tyr Ser AspIle Arg Ile Lys 435 440 445 Trp Thr Trp His Asn Val Leu Ser Arg Pro GlyAsn Asn Glu Cys Pro 450 455 460 Trp Gly His Ser Cys Pro Asp Gly Cys IleThr Gly Val Tyr Thr Asp 465 470 475 480 Ala Tyr Pro Leu Asn Pro Thr GlySer Ile Val Ser Ser Val Ile Leu 485 490 495 Asp Ser Gln Lys Ser Arg ValAsn Pro Val Ile Thr Tyr Ser Thr Ala 500 505 510 Thr Glu Arg Val Asn GluLeu Ala Ile Arg Asn Arg Thr Leu Ser Ala 515 520 525 Gly Tyr Thr Thr ThrSer Cys Ile Thr His Tyr Asn Lys Gly Tyr Cys 530 535 540 Phe His Ile ValGlu Ile Asn Gln Lys Ser Leu Asn Thr Leu Gln Pro 545 550 555 560 Met LeuPhe Lys Thr Glu Val Pro Lys Ser Cys Ser 565 570 1886 base pairs nucleicacid single linear DNA (genomic) unknown 5 ATGGAGTTGC CAATCCTCAAAGCAAATGCA ATTACCACAA TCCTCGCTGC AGTCACATTT 60 TGCTTTGCTT CTAGTCAAAACATCACTGAA GAATTTTATC AATCAACATG CAGTGCAGTT 120 AGCAAAGGCT ATCTTAGTGCTCTAAGAACT GGTTGGTATA CTAGTGTTAT AACTATAGAA 180 TTAAGTAATA TCAAGGAAAATAAGTGTAAT GGAACAGATG CTAAGGTAAA ATTGATGAAA 240 CAAGAATTAG ATAAATATAAAAATGCTGTA ACAGAATTGC AGTTGCTCAT GCAAAGCACA 300 CCAGCAGCAA ACAATCGAGCCAGAAGAGAA CTACCAAGGT TTATGAATTA TACACTCAAC 360 AATACCAAAA AAACCAATGTAACATTAAGC AAGAAAAGGA AAAGAAGATT TCTTGGTTTT 420 TTGTTAGGTG TTGGATCTGCAATCGCCAGT GGCATTGCTG TATCTAAGGT CCTGCACTTA 480 GAAGGAGAAG TGAACAAGATCAAAAGTGCT CTACTATCCA CAAACAAGGC CGTAGTCAGC 540 TTATCAAATG GAGTTAGTGTCTTAACCAGC AAAGTGTTAG ACCTCAAAAA CTATATAGAT 600 AAACAATTGT TACCTATTGTGAATAAGCAA AGCTGCAGAA TATCAAATAT AGAAACTGTG 660 ATAGAGTTCC AACAAAAGAACAACAGACTA CTAGAGATTA CCAGGGAATT TAGTGTTAAT 720 GCAGGTGTAA CTACACCTGTAAGCACTTAC ATGTTAACTA ATAGTGAATT ATTGTCATTA 780 ATCAATGATA TGCCTATAACAAATGATCAG AAAAAGTTAA TGTCCAACAA TGTTCAAATA 840 GTTAGACAGC AAAGTTACTCTATCATGTCC ATAATAAAAG AGGAAGTCTT AGCATATGTA 900 GTACAATTAC CACTATATGGTGTGATAGAT ACACCTTGTT GGAAATTACA CACATCCCCT 960 CTATGTACAA CCAACACAAAAGAAGGGTCA AACATCTGTT TAACAAGAAC TGACAGAGGA 1020 TGGTACTGTG ACAATGCAGGATCAGTATCT TTCTTCCCAC AAGCTGAAAC ATGTAAAGTT 1080 CAATCGAATC GAGTATTTTGTGACACAATG AACAGTTTAA CATTACCAAG TGAAGTAAAT 1140 CTCTGCAATG TTGACATATTCAATCCCAAA TATGATTGTA AAATTATGAC TTCAAAAACA 1200 GATGTAAGCA GCTCCGTTATCACATCTCTA GGAGCCATTG TGTCATGCTA TGGCAAAACT 1260 AAATGTACAG CATCCAATAAAAATCGTGGA ATCATAAAGA CATTTTCTAA CGGGTGTGAT 1320 TATGTATCAA ATAAAGGGGTGGACACTGTG TCTGTAGGTA ACACATTATA TTATGTAAAT 1380 AAGCAAGAAG GCAAAAGTCTCTATGTAAAA GGTGAACCAA TAATAAATTT CTATGACCCA 1440 TTAGTATTCC CCTCTGATGAATTTGATGCA TCAATATCTC AAGTCAATGA GAAGATTAAC 1500 CAGAGTTTAG CATTTATTCGTAAATCCGAT GAATTATTAC ATAATGTAAA TGCTGGTAAA 1560 TCAACCACAA ATATCATGATAACTACTATA ATTATAGTGA TTATAGTAAT ATTGTTATCA 1620 TTAATTGCTG TTGGACTGCTCCTATACTGT AAGGCCAGAA GCACACCAGT CACACTAAGC 1680 AAGGATCAAC TGAGTGGTATAAATAATATT GCATTTAGTA ACTGAATAAA AATAGCACCT 1740 AATCATGTTC TTACAATGGTTTACTATCTG CTCATAGACA ACCCATCTAT CATTGGATTT 1800 TCTTAAAATC TGAACTTCATCGAAACTCTT ATCTATAAAC CATCTCACTT ACACTATTTA 1860 AGTAGATTCC TAGTTTATAGTTATAT 1886 594 amino acids amino acid single linear DNA (genomic)unknown 6 Met Glu Leu Pro Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile LeuAla 1 5 10 15 Ala Val Thr Phe Cys Phe Ala Ser Ser Gln Asn Ile Thr GluGlu Phe 20 25 30 Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu SerAla Leu 35 40 45 Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu SerAsn Ile 50 55 60 Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val Lys LeuMet Lys 65 70 75 80 Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu LeuGln Leu Leu 85 90 95 Met Gln Ser Thr Pro Ala Ala Asn Asn Arg Ala Arg ArgGlu Leu Pro 100 105 110 Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys LysThr Asn Val Thr 115 120 125 Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu GlyPhe Leu Leu Gly Val 130 135 140 Gly Ser Ala Ile Ala Ser Gly Ile Ala ValSer Lys Val Leu His Leu 145 150 155 160 Glu Gly Glu Val Asn Lys Ile LysSer Ala Leu Leu Ser Thr Asn Lys 165 170 175 Ala Val Val Ser Leu Ser AsnGly Val Ser Val Leu Thr Ser Lys Val 180 185 190 Leu Asp Leu Lys Asn TyrIle Asp Lys Gln Leu Leu Pro Ile Val Asn 195 200 205 Lys Arg Ser Cys ArgIle Ser Asn Ile Glu Thr Val Ile Glu Phe Gln 210 215 220 His Lys Asn AsnArg Leu Leu Glu Ile Thr Arg Glu Phe Ser Val Asn 225 230 235 240 Ala GlyVal Thr Thr Pro Val Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255 LeuLeu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280285 Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290295 300 Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro305 310 315 320 Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile Cys LeuThr Arg 325 330 335 Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser ValSer Phe Phe 340 345 350 Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn ArgVal Phe Cys Asp 355 360 365 Thr Met Asn Ser Leu Thr Leu Pro Ser Glu ValAsn Leu Cys Asn Val 370 375 380 Asp Ile Phe Asn Pro Lys Tyr Asp Cys LysIle Met Thr Ser Lys Thr 385 390 395 400 Asp Val Ser Ser Ser Val Ile ThrSer Leu Gly Ala Ile Val Ser Cys 405 410 415 Tyr Gly Lys Thr Lys Cys ThrAla Ser Asn Lys Asn Arg Gly Ile Ile 420 425 430 Lys Thr Phe Ser Asn GlyCys Asp Tyr Val Ser Asn Lys Gly Val Asp 435 440 445 Thr Val Ser Val GlyAsn Thr Leu Tyr Tyr Val Asn Lys Gln Glu Gly 450 455 460 Lys Ser Leu TyrVal Lys Gly Glu Pro Ile Ile Asn Phe Tyr Asp Pro 465 470 475 480 Leu ValPhe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495 GluLys Ile Asn Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile 500 505 510Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys 515 520525 Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys Ser Thr Thr Asn 530535 540 Ile Met Ile Thr Thr Ile Ile Ile Glu Ile Ile Val Ile Leu Leu Ser545 550 555 560 Leu Ile Ala Val Gly Leu Leu Leu Tyr Cys Lys Ala Arg SerThr Pro 565 570 575 Val Thr Leu Ser Lys Asp Gln Leu Ser Gly Ile Asn AsnIle Ala Phe 580 585 590 Ser Asn 920 base pairs nucleic acid singlelinear DNA (genomic) unknown 7 TGCAAACATG TCCAAAAACA AGGACCAACGCACCGCTAAG ACACTAGAAA AGACCTGGGA 60 CACTCTCAAT CATTTATTAT TCATATCATCGGGCTTATAT AAGTTAAATC TTAAATCTGT 120 AGCACAAATC ACATTATCCA TTCTGGCAATGATAATCTCA ACTTCACTTA TAATTACAGC 180 CATCATATTC ATAGCCTCGG CAAACCACAAAGTCACACTA ACAACTGCAA TCATACAAGA 240 TGCAACAAGC CAGATCAAGA ACACAACCCCAACATACCTC ACTCAGGATC CTCAGCTTGG 300 AATCAGCTTC TCCAATCTGT CTGAAATTACATCACAAACC ACCACCATAC TAGCTTCAAC 360 AACACCAGGA GTCAAGTCAA ACCTGCAACCCACAACAGTC AAGACTAAAA ACACAACAAC 420 AACCCAAACA CAACCCAGCA AGCCCACTACAAAACAACGC CAAAACAAAC CACCAAACAA 480 ACCCAATAAT GATTTTCACT TCGAAGTGTTTAACTTTGTA CCCTGCAGCA TATGCAGCAA 540 CAATCCAACC TGCTGGGCTA TCTGCAAAAGAATACCAAAC AAAAAACCAG GAAAGAAAAC 600 CACCACCAAG CCTACAAAAA AACCAACCTTCAAGACAACC AAAAAAGATC TCAAACCTCA 660 AACCACTAAA CCAAAGGAAG TACCCACCACCAAGCCCACA GAAGAGCCAA CCATCAACAC 720 CACCAAAACA AACATCACAA CTACACTGCTCACCAACAAC ACCACAGGAA ATCCAAAACT 780 CACAAGTCAA ATGGAAACCT TCCACTCAACCTCCTCCGAA GGCAATCTAA GCCCTTCTCA 840 AGTCTCCACA ACATCCGAGC ACCCATCACAACCCTCATCT CCACCCAACA CAACACGCCA 900 GTAGTTATTA AAAAAAAAAA 920 298 aminoacids amino acid single linear DNA (genomic) unknown 8 Met Ser Lys AsnLys Asp Gln Arg Thr Ala Lys Thr Leu Glu Lys Thr 1 5 10 15 Trp Asp ThrLeu Asn His Leu Leu Phe Ile Ser Ser Gly Leu Tyr Lys 20 25 30 Leu Asn LeuLys Ser Val Ala Gln Ile Thr Leu Ser Ile Leu Ala Met 35 40 45 Ile Ile SerThr Ser Leu Ile Ile Thr Ala Ile Ile Phe Ile Ala Ser 50 55 60 Ala Asn HisLys Val Thr Leu Thr Thr Ala Ile Ile Gln Asp Ala Thr 65 70 75 80 Ser GlnIle Lys Asn Thr Thr Pro Thr Tyr Leu Thr Gln Asp Pro Gln 85 90 95 Leu GlyIle Ser Phe Ser Asn Leu Ser Glu Ile Thr Ser Gln Thr Thr 100 105 110 ThrIle Leu Ala Ser Thr Thr Pro Gly Val Lys Ser Asn Leu Gln Pro 115 120 125Thr Thr Val Lys Thr Lys Asn Thr Thr Thr Thr Gln Thr Gln Pro Ser 130 135140 Lys Pro Thr Thr Lys Gln Arg Gln Asn Lys Pro Pro Asn Lys Pro Asn 145150 155 160 Asn Asp Phe His Phe Glu Val Phe Asn Phe Val Pro Cys Ser IleCys 165 170 175 Ser Asn Asn Pro Thr Cys Trp Ala Ile Cys Lys Arg Ile ProAsn Lys 180 185 190 Lys Pro Gly Lys Lys Thr Thr Thr Lys Pro Thr Lys LysPro Thr Phe 195 200 205 Lys Thr Thr Lys Lys Asp Leu Lys Pro Gln Thr ThrLys Pro Lys Glu 210 215 220 Val Pro Thr Thr Lys Pro Thr Glu Glu Pro ThrIle Asn Thr Thr Lys 225 230 235 240 Thr Asn Ile Thr Thr Thr Leu Leu ThrAsn Asn Thr Thr Gly Asn Pro 245 250 255 Lys Leu Thr Ser Gln Met Glu ThrPhe His Ser Thr Ser Ser Glu Gly 260 265 270 Asn Leu Ser Pro Ser Gln ValSer Thr Thr Ser Glu His Pro Ser Gln 275 280 285 Pro Ser Ser Pro Pro AsnThr Thr Arg Gln 290 295 26 base pairs nucleic acid single linear DNA(genomic) unknown 9 ATCAATCAAA GGTCCTGTGA TAATAG 26 17 base pairsnucleic acid single linear DNA (genomic) unknown 10 CATGACTTGA TAATGAG17 86 base pairs nucleic acid single linear DNA (genomic) unknown 11AATTCATGGA GTTGCTAATC CTCAAAGCAA ATGCAATTAC CACAATCCTC ACTGCAGTCA 60CATTTTGTTT TGCTTCTGGT TCTAAG 86 27 base pairs nucleic acid single linearDNA (genomic) unknown 12 ACTGGCATCA ATCTAGCACT ACATGAG 27 136 base pairsnucleic acid single linear DNA (genomic) unknown 13 AATTCATGCCAACTTTAATA CTGCTAATTA TTACAACAAT GATTATGGCA TCTTCCTGCC 60 AAATAGATATCACAAAACTA CAGCATGTAG GTGTATTGGT CAACAGTCCC AAAGGGATGA 120 AGATATCACAAAACTT 136 94 base pairs nucleic acid single linear DNA (genomic)unknown 14 ATCATGGAGA TAATTAAAAT GATAACCATC TCGCAAATAA ATAAGTATTTTACTGTTTTC 60 GTAACAGTTT TGTAATAAAA AAACCTATAA ATAG 94 141 base pairsnucleic acid single linear DNA (genomic) unknown 15 ATCATGGAGATAATTAAAAT GATAACCATC TCGCAAATAA ATAAGTATTT TACTGTTTTC 60 GTAACAGTTTTGTAATAAAA AAACCTATAA ATATTCCGGA ATTCAGATCT GCAGCGGCCG 120 CTCCATCTAGAAGGTACCCG G 141 31 base pairs nucleic acid single linear DNA (genomic)unknown 16 CATGACTAAT TCCATCAAAA GTGAAAAGGC T 31 18 base pairs nucleicacid single linear DNA (genomic) unknown 17 CAAGAAAAAG GAATAAAA 18 39base pairs nucleic acid single linear DNA (genomic) unknown 18ATTTCTGTGA TATTTGTGCG GTATAATGAT GCTATACCT 39 18 base pairs nucleic acidsingle linear DNA (genomic) unknown 19 CAGGAGAAGG GTATCAAG 18 17 basepairs nucleic acid single linear DNA (genomic) unknown 20 AGGAGAAGGGTATCAAG 17 10 base pairs nucleic acid single linear DNA (genomic)unknown 21 AGGACAAAAG 10

What we claim is:
 1. A chimeric protein containing a protein ofparainfluenza virus (PIV-3) and a protein of respiratory syncytial virus(RSV) and selected from the group consisting of: (1) a chimeric proteincomprising a native PIV-3 F protein linked to a native RSV G protein;(2) a chimeric protein comprising a native PIV-3 F protein linked to anative RSV F protein; (3) a chimeric protein comprising a native PIV-3HN protein linked to a native RSV G protein; and (4) a chimeric proteincomprising a native PIV-3 HN protein linked to a native RSV F protein.2. The protein of claim 1 which is selected from the group consisting ofa native PIV-3 protein linked to a native RSV F protein(F_(PIV-3)-F_(RSV)), a native RSV F protein linked to a native PIV-3 HNprotein (F_(RSV)-HN_(PIV-3)) and a native RSV F protein linked to anative RSV G protein (F_(PIV-3)-G_(RSV)).
 3. A diagnostic reagent fordetecting infection by parainfluenza virus (PIV) and respiratorysyncytial virus (RSV) in a host, comprising the chimeric protein claimedin claim 1 and a carrier.